Veterinary pharmaceutical formulacion that comprises an RNA recombinant particle that encodes for a cu/zn superoxide dismutase protein of ruminant pathogenic bacteria and at least one RNA alphavirus belonging to the semliki forest virus family

a technology of rna and recombinant particles, which is applied in the field of veterinary pharmaceutical formulas, can solve the problems of not being able to protect i>brucella from patent application, affecting serological diagnosis, and not being able to work with sod protein in patent applications, so as to achieve high protection, protection efficacy, and high protection

Inactive Publication Date: 2011-08-18
UNIV DE CONCEPCION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0082]Table II shows the high efficacy of the expression systems to confer protection against the challenge from the pathogenic strain. The greatest protection efficacy against Brucella abortus occurred in mice immunized with rSFV4.2-SOD, where the presence of bacteria in the spleen was not observed. In addition, in the case of mice immunized with pSFV4.2-SOD, it was determined that a high level of protection was achieved.
[0083]This invention includes the development of two easy to use, highly efficient vaccines with a high level of biosecurity, whose response is greater than vaccines currently available on the market, such as the classic vaccines from attenuated organisms like strain RB51. The technology proposed is an alternative option for the development of one or more molecular vaccines against this bacterium.

Problems solved by technology

Among the strains mostly commonly used in attenuated vaccines is the vaccine whose active component uses strain 19 of Brucella abortus, but this vaccine causes abortions in the immunized animal and also develops antibodies against the O antigen of the LPS, interfering with the serological diagnosis of this disease.
The use of LPS as the active component for a possible vaccine has been tested, but it was observed that it offered no protection against Brucella sp.
However, the possibility exists that the plasmid DNA may be incorporated into the cell genome, for which reason its future use remains in doubt
This patent application does not work with the SOD protein.

Method used

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  • Veterinary pharmaceutical formulacion that comprises an RNA recombinant particle that encodes for a cu/zn superoxide dismutase protein of ruminant pathogenic bacteria and at least one RNA alphavirus belonging to the semliki forest virus family
  • Veterinary pharmaceutical formulacion that comprises an RNA recombinant particle that encodes for a cu/zn superoxide dismutase protein of ruminant pathogenic bacteria and at least one RNA alphavirus belonging to the semliki forest virus family
  • Veterinary pharmaceutical formulacion that comprises an RNA recombinant particle that encodes for a cu/zn superoxide dismutase protein of ruminant pathogenic bacteria and at least one RNA alphavirus belonging to the semliki forest virus family

Examples

Experimental program
Comparison scheme
Effect test

example 1

Extraction of Total Proteins from the Strain RB51 of Brucella abortus

[0088]The procedure for the extraction of bacteria included their culture. Once harvested, they were washed three times with sterile PBS at a pH of 7.2, centrifuged at 10000 rpm for 10 minutes at 4° C., eliminating the supernatant. The bacteria were inactivated in methanol at 60% for 24 hours. Subsequently the cells were washed again and kept for 24 hours at 4° C. in a hypertonic saline solution that contained NaCl (1 M), 0.1 sodium citrate and EDTA (0.5 mM). Then the cells were sonicated for approximately 15 minutes at 60 watts, and centrifuged at 10000 rpm for 10 minutes at 4° C. 0.2 mM of phenylmethylsulfonyl fluoride (PMSF) were added to the supernatant and the proteins were concentrated with polyethylenglycol in dialysis bags with a molecular weight retention capacity above 3500 kD. Then the fraction concentrated in this way was dialyzed against distilled water for two days, at the end of which it was centrif...

example 2

Expression of the Recombinant Cu / Zn Superoxide Dismutase Protein (see FIG. 6)

[0089]The Cu / Zn SOD protein of Brucella abortus was expressed in E. coli DH5 bacteria that were transformed by electroporation with the plasmid pBSSOD that contains the gene that encodes the Cu / Zn SOD protein (sodC). To obtain the protein the bacterium was cultured in LB broth plus 100 μg / ml of ampicillin for 12 hours at 37° C., with agitation. Subsequently the bacteria were collected from the culture broth and centrifuged at 3000 rpm for 20 minutes. The bacteria were re-suspended in 10 mM phosphate buffer at pH 7.6 plus 0.1% of Triton X-100 and were incubated at 37° C. for 12 hours, with agitation. The mixture was centrifuged at 10000 rpm for 20 minutes, and the recovered supernatant was then added to a column of anionic exchange, which has no affinity for the Cu / Zn SOD protein, most of the other proteins present in the supernatant being retained. The eluate obtained from the column was treated with polymi...

example 3

Construction of the Plasmid

[0090]Once the original gene of the Cu—Zn superoxide dismutase protein (SodC) had been obtained using restriction enzymes from the plasmid pUC19, the plasmid pSFV4.2-SOD was constructed. For this purpose, the plasmid PUC19 was subjected to digestion with the enzymes BamHI and XhoI for 2 hours at 37° C. From the digestion a fragment of 1.1 kb was obtained that contained the gene of interest, which was extracted from 1% agarose gel using a commercial kit. (see FIG. 4, Line 2). In addition, the plasmid pSFV4.2 was digested with the same restriction enzymes used previously and in the same conditions. At the end of incubation, the restriction enzymes were inactivated at 60° C. for 15 minutes. Subsequently, ligation was carried out, mixing in a proportion of 3:1 the 1.1 kb insert with the plasmid pSFV4.2, which presented a marker for the ampicillin antibiotic. This was previously digested using the ligase enzyme DNA T4 in ligase buffer DNA T4 10× plus 5 mM of AT...

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Abstract

The technology is a veterinary pharmaceutical formulation of two vaccines, one from an RNA viral vector system constituted by an RNA recombinant particle that codifies for a Cu/Zn superoxide dismutase protein of Brucella abortus, and the other based on naked RNA constituted by a recombinant molecule of naked RNA that carries a sequence for the synthesis of at least one recombinant Cu/Zn superoxide dismutase protein of Brucella abortus and some Semliki Forest virus genes. An expression system based on the Semliki Forest virus and a use of this system, in addition to a method for the preparation of the pharmaceutical formulations.

Description

[0001]This technology is designed for the stockbreeding sector, specifically for bovines, which have a high rate of abortions caused by the bacterium Brucella abortus. Previous Techniques[0002]Brucella abortus is a facultative, intracellular gram-negative bacterium that contains mannose molecules facilitating adherence to the mononuclear phagocytes of the host. In particular, the bovine placenta contains a great number of mannose receptors, which is favor the internalization of this bacterium and consequently the probability of abortions in these animals[0003]When the macrophage recognizes conservation patterns on the surface of Brucella sp. (LPS or external membrane proteins) it is activated and then phagocytizes the bacterium. However, Brucella sp. survives very efficiently within the phagocytic cell, since it is able to avoid the fusion of the lysosome with the phagosome. Brucella sp. avoids the respiratory burst inside the phagolysosome, since it avoids the formation of oxygen d...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K9/127A61K31/7088A61P31/04
CPCA61K38/00A61K39/098A61K2039/5256C12N2770/36143A61K2039/552A61K2039/55555C12N9/0089A61K2039/53A61P31/04A61P37/04
Inventor ONATE CONTRERAS, ANGELANDREWS GARCIA, EDILIADONOSO NANCULAO, GABRIEL
Owner UNIV DE CONCEPCION
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